Glycoconjugates such as glycoproteins, proteoglycans, or glycolipids are important biological materials, which are produced by bonding protein or lipid with a carbohydrate chain that may be of various kinds. One group of the glycoconjugates, glycoproteins, is broadly classified into serotype glycoproteins and mucin glycoproteins.
In most of proteins in serum excluding albumin, carbohydrate is linked to an Asn of Asn-X-Ser/Thr residue in the proteins through N-linkage. Thus, a glycoprotein having a linkage of this type is termed as serum-type glycoprotein or N-linked glycoprotein. Further, a carbohydrate chain, which is linked to the N-linked glycoprotein, is termed as an N-linked carbohydrate chain.
On the other hand, in protein being a main component of: (i) mucus secreted from a glandular system such as a salivary gland or submandibular gland; and (ii) a mucous tissue of an inner surface of a gastrointestinal tract of a stomach, a small intestine or the like, carbohydrate is linked to Ser/Thr through O-linkage. Therefore, a glycoprotein having a linkage of this type is termed as mucin glycoprotein or O-linked glycoprotein. Further, a carbohydrate chain bonded to the O-linked glycoprotein is termed as an O-linked carbohydrate chain.
In vivo, there are various kinds of carbohydrate chains. Some proteins such as IgG, IgE, and the like, contain the N-linked carbohydrate chain. Some proteins such as submandibular gland mucin and the like contain the O-linked carbohydrate chain. Further, some proteins such as erythropoietin and the like contain both the N-linked carbohydrate chain and the O-linked carbohydrate chain.
These glycoproteins play various and essential roles in vivo. For example, a carbohydrate chain of IgG causes protein to retain its steric structure, mucin performs defense effect against xenobiotic substances, erythropoietin adjusts its metabolic rate in blood, and asialoglycoprotein has a function of being uptaken to a liver and metabolized therein. Further, it is considered that these carbohydrate chains are widely found on a surface of a cell membrane and in intercellular matrix among cells, and plays a part of essential roles in biological information network. However, details of the function of the carbohydrate chains in the biological information network have not been fully understood.
As described above, the carbohydrate chains in glycoproteins have various functions. In order to understand the functions, structural analysis should be carried out after separation of a sugar chain from a glycoprotein to which it is attached. For the structural analysis, the carbohydrate chain is cleaved from protein and then separated. With recent improvement of analyzers and methods for structural analysis, separation of a carbohydrate chain derived from glycoprotein and its structural analysis can be carried out by sophisticated separating means such as high-performance liquid chromatography and capillary electrophoresis. Further, the followings have facilitated structural analysis of a separated carbohydrate chain: (i) molecular weight measurement by a matrix-assisted laser desorption time of flight mass spectrometry or electrospray mass spectrometry; (ii) sequence determination by fragment ion analysis; and (iii) structural analysis by high-field nuclear magnetic resonance.
By fully utilizing the leading-edge analyzers described above, various methods have been realized for structural analysis, though there are still some difficulties, especially, in obtaining a required amount of carbohydrate chains separated from glycoprotein.
On the other hand, many researchers have been trying to develop a simple method for cleaving a carbohydrate chain from glycoprotein. Regardless of many studies, no simple method has been found. Therefore, in the carbohydrate chain analysis, the cleavage of the carbohydrate chain still requires experience. Further, in the analysis of a carbohydrate chain, the cleavage of the carbohydrate chain is still rate-determining step.
For example, in order to cleave an N-linked carbohydrate chain off from an N-linked glycoprotein linked to asparagine, a chemical method and an enzymatic method have been mainly used.
Specifically, hydrazinolysis has been used as a typical chemical method. In the hydrazinolysis method, a carbohydrate chain is separated by heating, at high temperature (100° C. or higher) for long hours, a solution in which a subject glycoprotein is dissolved in anhydrous hydrazine. As is generally known, however, anhydrous hydrazine is highly toxic and explosive and thus requires special care for handling. There are several methods for separating a carbohydrate chain from a glycoprotein with the use of hydrazine, and all of the methods require multiple steps: (i) pyrogenetic reaction for 3 to 24 hours; (ii) removal of hydrazine; (iii) re-acetylation of an amino group of amino carbohydrate in a carbohydrate chain; and (iv) its post-treatment. In order to complete the entire process, at least two days (48 hours) or more are required.
In the enzymatic method which is another method for separating an N-linked carbohydrate chain from an N-linked glycoprotein, a carbohydrate chain is separated by utilizing N-glycanase F found in a microorganism of flavobacterium spp. or N-glycoamidase A found in almond seeds. As a substrate, glycopeptide obtained by digestion with protease (e.g. trypsin) is often employed. Unlike the hydrazinolysis method, the enzymatic method is safe. However, the enzymatic method requires experiences due to the use of enzyme, and takes one day (24 hours) or more for achieving general enzymatic reaction. Further, as to N-glycosidase F, though recombinant products are commercially available, only a slight amount (less than milligram) is generally used due to its high price. More seriously, depending on a molecule size of enzyme as protein, some carbohydrate chains are not easily separated from glycoproteins or not separated at all. Due to such a drawback that enzymatic functions differ depending on kinds of glycoproteins, enzymatic reaction cannot be employed directly to cells and body fluids.
On the other hand, the chemical method, but not the enzymatic method, is used for separating a carbohydrate chain from an O-linked glycoprotein linked to a hydroxyl group of serine or threonine in a glycoprotein. The enzymatic method is not used, because an O-linked carbohydrate chain having eight kinds of core structures and no enzyme has been found that has a diverse spectrum that enables recognition of all of the eight kinds of core structures.
Thus, in order to separate an O-linked carbohydrate chain from an O-linked glycoprotein, an alkali decomposition method has been exclusively used. The alkali decomposition method takes an advantage of the nature of alkali, which facilitates cleaving a linkage between the O-linked carbohydrate chain and an Ser or Thr. In the alkali decomposition method, a carbohydrate chain is separated by dissolving O-linked glycoprotein in a aqueous solution at high concentration of alkali, and incubating it for long hours (generally 48 hours or longer). As to the aqueous alkali solution, sodium hydroxide of 0.1 to 0.5 M is generally used. Due to its high concentration, however, there is a fatal problem that a carbohydrate chain separated from a glycoprotein is decomposed through β-elimination reaction. The β-elimination reaction occurs due to C-1 position hemiacetal group of the carbohydrate chain cleaved from the glycoprotein.
In order to solve the problem, the alkali decomposition method is arranged such that the carbohydrate chain cleavage reaction is carried out using the alkaline solution to which a reducing agent (e.g. NaBH4) is added. However, this causes its post treatment to be more complicated because the reducing terminal of the carbohydrate chain are not a hemiacetal structure and reduced to sugar alcohol, not a hemiacetal structure. As another drawback, the sugar alcohol cannot be labeled with a fluorogenic or chromogenic reagent, which is necessary for highly sensitive analysis. Therefore, the hydrazinolysis method has been used as an alternative to the alkali decomposition method. Since the hydrazinolysis method not only requires long hours for reaction and post processing, but also utilizes an highly explosive agent, the hydrazinolysis method is poor in versatility.
Recently, such methods have been reported that efficiently cleave an O-linked carbohydrate chain from O-linked glycoprotein without modifying a reducing terminal. In the methods, ammonia is used for cleaving a carbohydrate chain (Huang, Y. ; Mechref, Y. ; Novotny, M. V., 2001, Microscale nonreductive release of O-glycans for subsequent analysis through MALDI mass spectrometry and capillary electrophoresis., Anal. Chem., vol 73,6063-6069), or an O-linked glycoprotein is adsorbed in a column to separate an O-linked carbohydrate chain (Karlson, N. G.; Packer, N. H., 2002, Analysis of O-linked reducing oligosaccharides released by an in-line flow system., Anal. Biochem., vol 305,173-185).
However, the both conventional methods have to be carried out under mild condition for long hours in order to prevent a carbohydrate chain from being decomposed, because a cleaved O-linked carbohydrate chain goes through β-elimination reaction. Thus, there will be great difficulties in applying the conventional methods to clinical analysis which requires a large number of samples, or to proteome analysis or proteomics analysis which requires high throughput.
As described above, studies on the functions of carbohydrate chains have been proceeded with difficulties. One of the difficulties come from diverse structures of carbohydrate chains, in which each monosaccharide, which is component of the carbohydrate chain, has four to five hydroxyl groups respectively. However, the biggest difficulty comes from the fact that a simple method has not been found for readily cleaving a carbohydrate chain off from glycoconjugate. Specifically, as to glycoprotein, which is a conjugation of protein and a carbohydrate chain(s) with molecular weight of several ten thousands to several hundred thousands, a method for cleaving off a carbohydrate chain has been searched for thirty years. However, no excellent versatile method has been found.
Consequently, there has been a strong demand for realizing a method for separating a carbohydrate chain from glycoprotein readily and securely in a short time.
The present invention was made in view of the above problem, and an object of the present invention is to provide: (i) a method for readily separating carbohydrate in a short time by efficiently cleaving a glycoside linkage; (ii) a carbohydrate separating system for applying the method to clinical analysis or to proteome analysis or proteomics analysis which requires high throughput; (iii) a carbohydrate separating reagent kit; (iv) a standard sample for carbohydrate separation ; and (v) an evaluation method and an evaluation system for carbohydrate.